UV-VIS Spectrophotometry A Brief Background to Spectrophotometry Contents Electromagnetic Spectrum Introduction ....................................................... 1 The electromagnetic spectrum ranges from Electromagnetic Spectrum............................... 1 Gamma radiation, with the smallest wavelength (1 Radiation and the Atom .................................... 2 pm), to Low Frequency radiation, with the largest Radiation and the Molecule.............................. 2 wavelength well beyond conventional radio waves Electron Transitions ....................................... 2 (100 Mm or 100000 km). Human beings can only Vibration and Rotation ................................... 4 directly detect a very small portion of this Specific Absorption .......................................... 4 spectrum, with thermal perception of radiant heat Absorption and Concentration ........................ 5 being a sensitivity to infrared (IR) radiation, and Instrumentation ................................................. 6 sight is limited to the VIS spectrum. The spectrum Light Source ................................................... 6 is smoothly continuous and the labelling and Monochromator .............................................. 7 assignment of separate ranges are appointed Optical Geometry ........................................... 8 largely as matter of convenience (Figure 1). UV- Sample Handling .......................................... 10 VIS spectrophotometry concerns the UV range Detectors ...................................................... 10 covering of 200-380 nm and the VIS range Measuring Systems ..................................... 11 covering 380-770 nm. Many instruments will offer Good Operating Practice ................................ 11 slightly broader range from 190 nm in the UV Limitations of Beer-Lambert Law ................. 12 region up to 1100 nm in the near infrared (NIR) Sources of Error .............................................. 12 region. Instrument Sources of Error ......................... 12 Non-instrument Sources of Error ................. 14 All electromagnetic radiation travels at the speed Bibliography .................................................... 14 of light in a vacuum (푐), which equals 3×108 m/s, Contact Us ....................................................... 15 the distance between two peaks along the line of travel is the wavelength, (휆), and the number of Introduction peaks passing a point per unit time is the frequency (푣). The mathematical relationship The spectrophotometer is ubiquitous among between these three quantities is expressed modern laboratories. Ultraviolet (UV) and Visible using: (VIS) spectrophotometry has become the method of choice in most laboratories concerned with the 푐 = 휆휈 identification and quantification of organic and inorganic compounds across a wide range of Additionally, the laws of quantum mechanics products and processes. Applied across research, defines the energy of a single particle of light, a quality, and manufacturing, with continuing focus photon, as: on life science and pharmaceutical environments, they are equally as relevant in agriculture, animal 퐸 = ℎ휈 husbandry and fishery, geological exploration, food safety, environmental monitoring, and many Where 퐸 is the energy of the radiation, and ℎ is manufacturing industries to name a few. Planck's constant. Combining these two equations gives: Modern spectrophotometers are quick, accurate, and reliable. They require only small demands on 퐸 = ℎ푐/휆 the time and skills of the operator. However, the non-specialised end-user who wants to optimise Which shows that the energy is inversely the functions of their instrument, and be able to proportional to wavelength. That is to say that the monitor its performance will benefit from the shorter the wavelength the higher the energy. appreciation of the elementary physical laws governing spectrophotometry, as well as the basic In the visible region it is convenient, and the elements of spectrophotometer design. This brief modern convention, to define the wavelength in background to spectrophotometry offers an nm (nanometres), which is 10-9 m. However, insight to support users of Biochrom’s range of spectrophotometers. 1 Author: Luke Evans, PhD. Technical Support and Application Specialist at Biochrom Ltd. Issue 1.0 Figure 1: The illustration describes the electromagnetic spectrum with the visible (VIS) range expanded for further subdivision. historical literature may display alternative units, Bohr model to explain the electronic phenomena such as millimicron (mμ) or Angstrom (Å). These which concerns spectrophotometry. are simply converted using: The Rutherford–Bohr model defines an atom as 1 푛푚 = 1 푚µ = 10 Å having a number of electron shells, n1, n2, n3 and so on, in which the increasing values of n represent higher energy levels and greater Radiation and the Atom distance from the nucleus. Electrons orbit the nucleus in subshells, designated s, p, d, and f, It is convenient to describe electromagnetic within each shell. Each n-shell contains a radiation as waves. However to clearly configuration of s, p, d, and f subshells and each demonstrate the interactions that lead to specific subshell can house a maximum of two electrons absorption, it is helpful to consider the radiation as (Figure 2). No two electrons can have identical discrete packages of energy, or quanta, called energies, but for succinctness they can be photons. grouped related to the n-shell they occupy, 1s, 2s, 2p, 3s, 3p, 3d, and so on. The phenomena of absorbance depends upon the atomic structure, more specifically the atomic By considering atoms of sodium vapour, the effect orbitals which each of the electrons of those of subjecting an atom to an appropriate radiation atoms occupy and the associated energy levels of can be demonstrated. Excitation of an electron, in those orbitals. Occupied orbitals are finite and well the outermost subshell of a sodium atom, by a defined, but an electron can be moved to a more photon at 589, 330 or 285 nm will promote its energetic orbital, in a process called electron transition to varying excited states; corresponding excitation, providing a quantum of energy equal to with the higher energy (shorter wavelength) of the the energy difference between the ground and radiation (Figure 3.). excited state is delivered. Excited states are generally unstable and the electron will rapidly revert to the ground state, in a process termed Radiation and the Molecule electron relaxation, losing the acquired energy, described as emission. Electron Transitions Whilst the accepted model of atomic and Electrons in the atom can be considered as molecular structure has arisen from the occupying groups of similar energy levels. The Schrödinger wave mechanical treatment, it is more complicated molecular model shows convenient to employ the simpler Rutherford– bonding electrons associated with more than one 2 Figure 2: The illustrations above show the spatial geometries of atomic Subshells subshells. Each electron pair can occupy any space of the same colour, allowing for two pairs in each geometry except for the s subshell. They are s p d f shown on xyz axes and the specific nomenclature, based on their orientation n1 (2) 1 (2) about that axis, is shown below each illustration. Within larger atoms subsequent subshells envelope the equivalent subshell of the previous n- n2 (8) 1 (2) 3 (6) shell, but maintain the same geometry. The table left shown the subshell Shell n3 (18) 1 (2) 3 (6) 5 (10) composition of the first four n-shells. The number in each cell defined the number of that given subshell at that n-shell level, and the numbers in n4 (32) 1 (2) 3 (6) 5 (10) 7 (14) brackets define the maximum number of electrons that can be accommodated in each shell and subshell. nucleus, and are particularly susceptible to transitions from bonding MO’s to their relative subshell transitions. antibonding MO’s, and from non-bonding MO’s to either antibonding MO’s (Figure 4). The electrons concerned, may be present in one of two chemical bond types; sigma (σ) bonds which result from s-subshell overlap, or the generally weaker pi (π) bond which results from p- subshell overlap. Figure 4: The diagram shows the electron transitions between molecular orbital (MO) types. σ and π-bonds without an asterisk denote bonding MO’s, while those marked with an asterisk denote antibonding MO’s, and n denotes a non-bonding MO. Due to relatively high stability of σ-bonds, the σ → σ* and n → σ* transitions require relatively high energy, and are therefore associated with shorter wavelength radiation UV. Whereas the relatively low stability of the π-bond, means that n → π* and π → π* are associated with both UV and larger wavelength VIS radiation. The presence of a carbon-carbon double bond in the molecule increases the likelihood of π-bonds. Especially if they alternate with single bonds (conjugate double bonds), where one of the Figure 3: The diagram illustrates the energy delivered bonding MO’s is raised in energy and the other resulting in alternative subshell transitions. lowered relative to the energy of an isolated double bond. The same applies to the antibonding Chemical bonds are formed by overlapping MO’s. The effect is greater still if the bond contains atomic shells that result in one of three types of a highly electronegative atom, such as nitrogen, Molecular Orbital (MO); bonding (low energy), which attract electrons more strongly. As a result, antibonding